Myoglobin and Hemoglobin BL4010 10.12.06

Myoglobin & Hemoglobin Objectives Identify biological functions. Identify parts of Mb & their roles in O2 transport. Identify how Hb differs from Mb.

Myoglobin O2 transport, storage in cells. Two parts: protein and heme prosthetic group. Protein: 155 amino acids, ~ 17 kDa. Compact, globin fold. 75% helix.

Heme Heme is composed of porphyrin and Fe2+. Porphyrin is non-polar, except two proprionate groups. Porphyrin binds O2, CO. CO 10,000x tighter than O2.

Heme Ligands Fe is 6-coordinate. Four N of heme group One N from proximal His. One from H2O or O2. Can also bind CO.

Heme – Protein Interaction carboxyl group of proprionate. Heme stabilizes protein fold. Binds through hf interactions Prooprionate groups on surface.

Myoglobin Protein completely surrounds heme. Function of protein: ↑ heme solubility. ↓ heme oxidation (Fe2+ → Fe3+) metmyoglobin inactive. ↓ CO binding.

O2 vs. CO binding. CO binds tightly; linear. Fe C O CO binds tightly; linear. O2 binds less tightly, bent structure. Distal His forces bent binding of both, weakens CO binding. Distal His Proximal His

Hemoglobin Tetrameric protein Dimer of dimers, (ab)2 a,b chains resemble Mb. Each chain contains, heme, prox. histidine. Each binds 1 eq. O2. ab dimer interface different from aa, bb interface. Marked by salt bridges that stabilize the deoxy structure.

Oxygen Binding Curves Objectives. Understand O2 binding curve for Mb. Understand O2 binding curve for Hb. Identify mechanism of cooperative binding.

Y = [MbO2] / ([Mb] + [MbO2]) Quantify O2 binding. Measure deoxy- vs. oxy by visible absorption (Soret band). Reaction is Mb + O2 MbO2 Equil. Const. given by: Ka = [MbO2]/[Mb][O2] Plot fraction bound: Y = [MbO2] / ([Mb] + [MbO2]) Recast in terms of measureable quantities: Y = pO2 / (pO2 + pO2,50)

Direct Plots Plot fractional saturation versus partial pressure of O2. Most relevant part of plot in range of cell pO2 (~ 15-25 mm Hg).

Direct Plot Binding characterized by pO2,50. Partial pressure of O2 where Mb is half saturated. If lower oxygen affinity, curve shifts right; if higher, curve shifts left. pO2,50 = 3 mm Hg.

4° Structure of Hb alters O2 binding. Interactions between dimers alters oxygen binding. Direct plot shows Hb has lower affinity than Mb. Sets up delivery system. O2 bound by Hb in lungs; released in tissues. Mb Hb

Cooperative regulation Hb oxygen binding: Start binding with given affinity in deoxy state, subsequent binding enhances affinity. Defines positive cooperative regulation. Only seen in multi-domain proteins. Hill coefficient ~ number of interacting subunits. Advantage: binding is more sensitive to small changes in [ligand].

Molecular Model In deoxy state, Fe out of heme plane; domed. Bind O2, moves Fe. This moves proximal His and its helix. Moving helix alters a/b interface. Deoxy = Tense (T) Oxy = Relaxed (R) T R

T → R Structural Changes. T form: H143 - D94 in b1. K40 - H146 CO2-. R141 - D126 a2/a1. R form: All are broken. deoxy oxy

Small changes translate to large movements. Deoxy State

Small changes translate to large movements. Oxy state

Modifying O2 Binding in Hb Objectives. Identify allosteric effectors Describe molecular basis of each.

Bohr Effect Oxygen affinity sensitive to pH. ↓ pH; ↑ pO2,50 (lowers sensitivity). D94 ↔ H146 salt bridge in T state only. Excess H+ forms salt bridge, favors deoxy state. log pO2 log Q H146 D94

CO2 Produced during aerobic metabolism. log pO2 log Q Produced during aerobic metabolism. Reacts with N terminal amino; carbamylation reaction. Negative charge forms salt bridge with aR141, stabilizes deoxy state. R-NH2 + CO2 R-NH-CO2- + H+ In tissues, High CO2 In lungs, Low CO2

CO2 is Coupled to Bohr Effect In Tissue CO2 is bound by Hb or converted to bicarbonate by carbonic anhydrase. Buffers blood pH. Hb binds 2H+ / 4 O2 released, also buffers (Bohr effect). In Lungs Low pCO2; reaction reverses; CO2 and H+ released from Hb, pO2,50 decreased (increased oxygen affinity). From Lange’s Biochemistry

Chloride Anion Binding Also favors T state Forms salt bridge with R141, V1 in T state. Released in R state.

2,3-BPG Side product of glycolysis. indicates active respiration, need O2. Binds cationic region in T-form. Favors deoxy, releases O2 to tissues. [2,3-BPG] is high, responsible for observed pO2,50 of 27 mm Hg. stripped Hb has pO2, 50 ~ 8 mm Hg.

Another look at 2,3-BPG BPG acts as a “wedge” and drives the R state to the T state. Forces release of bound O2 in active tissue. BPG increases at high altitude.

Arginine → Citrulline + NO NO and Hb NO potent vasodilator Produced by Nitric Oxide Synthase (NOS). Arginine → Citrulline + NO Activates soluble guanylyl cyclase, signal transduction cascade. Reacts with Hb, which inactivates the NO (can’t react with sGC). Interactions with Hb Binds to HbO2 to form nitrates (NO3-) Binds to deoxy-Hb to form iron-nitrosyl (Fe2+-NO). Rapid reaction in vitro, but slow in vivo due to nature of blood flow along endothelium and diffusional barriers.

Transport of NO by Hb NO also binds to Cys93 on b-chains. Forms S-NO bond (SNO-Hb). Transfers NO to glutathione, which functions as a storage form for NO (won’t react with HbO2).

NO transport by Hb Transfer reaction depends on T/R state. R-state promotes binding to Cys93 SNO-Hb formed in lungs. NO released in T-state. Effectively delivers NO to vasculature. Vasodilation then enhances O2 delivery. But wait a minute … [SNO-Hb] 10,000 x lower than HbO2. NO transfer from SNO-Hb is slow. Amount released can’t compete with NO produced by NOS in erythrocytes.

Clinical relevance If NOS impaired, SNO-Hb is effective transport, O2 delivery system. Therapeutic value in treatment of sickle cell? Use in blood substitutes?